Tailor-made transcriptional biosensors for optimizing ... · Tailor-made transcriptional biosensors...
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Tailor-made transcriptional biosensors for optimizing microbial cell factories Brecht De Paepe 1 , Jo Maertens 1 , Bartel Vanholme 2 and Marjan De Mey 1 1 CSB - Centre for Synthetic Biology - Ghent University - Belgium 2 VIB - Vlaams Instituut voor Biotechnologie - Department of Plant Systems Biology - Belgium Centre for Synthetic Biology pNatSens2&27haracterization2of2native2biosensor2circuit pSynSens2&2Wecoupling2of2the2detector2and2effector2module pSynSens)OX2&2àngineering2the2detector2module pSynSensPOX2&2àngineering2the2effector2module22 pNatSens Introduction Monitoring2the2intra&2and2extracellular2environment2and2adapting2metabolic2processes2accordingly2is2key2to2properly2address2the2enormous2complexity2of2today’s2metabolic2engineering2questionsO2To2 this2endA2scientists2are2reprogramming2nature’s2myriad2of2transcriptional2regulatory2systems2into2transcriptional2biosensors2which2are2able2to2detect2small2molecules2andA2in2responseA2express2output2 signalsO2In2this2mannerA2biosensors2propel2key2applicationsA2such2as2high&throughput2screeningA2adaptive2laboratory2evolution2and2dynamic2pathway2controlO2HenceA2transcriptional2biosensors2bear2 the2potential2to2augment2and2accelerate2current2metabolic2engineering2strategiesA2catalyzing2vital2advances2in2industrial2biotechnologyO2HoweverA2the2two2key2characteristics2of2these2natural2biosensor2 circuitsA2the2response2curve2and2ligand2specificityA2are2typically2not2in2line2with2the2requirements2for2a2real&life2biosensor2applicationO2InsteadA2they2are2evolutionary2optimized2for2specific2in2vivo2 conditionsO27onsequentlyA2engineering2efforts2are2imperative2to2generate2tailor&made2biosensors2witch2custom2response2curves2and2ligand2specificitiesO2HenceA2in2this2contribution2to2address2these2 limitations2of2natural2biosensorsA2several2engineering2principles2were2established2to2create2biosensors2with2custom2response2curvesO2 The2authors2wish2to2thank2the2M8gentschap2Innoveren2q2OndernemenM2in2Klanders2,8IOw2for2financial2 support2in2the2framework2of2the2PhOWO2grant2of2/recht2We2PaepeO2This2research2is2also2supported2by2the2 7ondex2project2,GO(BP)O)BNwO2 /recht2We2Paepe2&2/rechtOWePaepe4UGentObe 7entre2for2Synthetic2/iology2&2Ghent2University 7oupure2Links20GBA2U(((2Ghent2&2/elgium2 Wetector2module àffector2module Transcription factor Fluorescent protein Ligand2molecule Kluorescent2protein Transcription2factor Transcription2factor2binding2sites Promoter R/S 7oding2sequence pSynSens 8cknowledgments 7ontact Wetector2module àffector2module R/S2&2Promoter2&2Spacer2&2Terminator ? Kitted2Hill2function2of2pNatSens Response2curve2of2pNatSens P( ( j( 0( @( )(( Ligand2concentration2,mgFlw KluorescenceFoptical2density2,&w P( ( j( 0( @( )(( Kitted2Hill2function2of2pSynSens Response2curve2of2pSynSens P((( ( j((( )((( B((( G((( P((( ( j((( )((( B((( G((( 0((( 3((( 5'UTR 5'UTR With2 the2 different2 genetic2 parts2 identifiedA2 the2 path2 is2 paved2to2engineer2this2biosensor2circuit2by2altering2the2 expression2of2the2detector2and2effector2moduleA2vis&à& vis2each2otherO2HoweverA2both2modules2are2entwined2in2 this2bidirectional2intergenic2architecture2and2can2not2be2 altered2 without2 affecting2 the2 opposite2 moduleO2 ThereforeA2the2two2modules2are2decoupled2by2inserting2 a2 terminator2 and2 spacer2 sequence2 together2 with2 a2 defined2 promoter2 and2 R/S2 sequence2 to2 constitutively2 express2 the2 transcription2 factorO2 This2 synthetic2 biosensor2 circuitA2 iOeO2 pSynSensA2 was2 constructed2 without2loss2of2functionality2in2comparison2to2pNatSensO /y2varying2the2R/S2strength2of2the2detector2moduleA2a2set2of2custom2biosensors2was2created2with2 differing2 characteristics2 such2 asA2 increased2 operational2 and2 dynamic2 rangeA2 varying2 sensitivity2 and2cooperativityA2resulting2in2distinct2Hill2parametersO2NoteworthyA2when2targeting2the2detector2 moduleA2 specific2 R/S2 strengths2 resulted2 in2 increased2 output2 variability2 which2 is2 atributed2 to2 unbalanced2levels2of2transcription2factor2in2relation2to2the2regulatory2mechanismO2 8kin2to2the2detector2moduleA2varying2the2R/S2strength2of2the2effector2module2resulted2in2a2set2of2 custom2 biosensors2 with2 differing2 characteristicsO2 NoteworthyA2 when2 targeting2 the2 effector2 moduleA2 a2 wider2 range2 of2 maximum2 response2 levelsA2 with2 twofold2 higher2 maximum2 response2 levelsA2was2achieved2in2comparison2to2engineering2the2detector2moduleO Ligand2concentration2,mgFlw KluorescenceFoptical2density2,&w P( ( j( 0( @( )(( ( P((( j((( 0((( @((( )(((( )P((( pSynSens Fit pSS1_RBS = 10 (T.I.R.) pSS1_RBS = 100 (T.I.R.) pSS1_RBS = 500 (T.I.R.) pSS1_RBS = 1000 (T.I.R.) pSS1_RBS = 2500(T.I.R.) pSS1_RBS = 5000 (T.I.R.) pSS1_RBS = 20000 (T.I.R.) 0 5000 10000 15000 20000 25000 0 20 40 60 80 100 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 k2&2Maximum2response2level2,&w K M 2&2Hill2constant2,mgFlw n2&2Hill2coefficient2,&w pSynSens )(( G(( )((( PG(( G((( P(((( pSynSens )(( G(( )((( PG(( G((( P(((( pSynSens )(( G(( )((( PG(( G((( P(((( )(((( )G((( PG((( R/S2strength2,TOIOROw R/S2strength2,TOIORO2&2translation2initiation2ratew R/S2strength2,TOIOROw pSynSens )(( G(( )((( PG(( G((( P(((( R/S2strength2,TOIOROw R/S2strength2,TOIORO2&2translation2initiation2ratew R/S2strength2,TOIOROw 25000 0 5000 10000 15000 20000 k2&2Maximum2response2level2,&w 0 20 40 60 80 100 pSynSens )(( G(( )((( PG(( G((( P(((( )(((( )G((( PG((( K M 2&2Hill2constant2,mgFlw 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 pSynSens )(( G(( )((( PG(( G((( P(((( )(((( )G((( PG((( n2&2Hill2coefficient2,&w Ligand2concentration2,mgFlw P( KluorescenceFoptical2density2,&w ( G((( )(((( )G((( P(((( pSynSens Fit pSS2_RBS = 10 (T.I.R.) pSS2_RBS = 100 (T.I.R.) pSS2_RBS = 500 (T.I.R.) pSS2_RBS = 1000 (T.I.R.) pSS2_RBS = 2500 (T.I.R.) pSS2_RBS = 5000 (T.I.R.) pSS2_RBS = 10000 (T.I.R.) pSS2_RBS = 15000 (T.I.R.) pSS2_RBS = 20000 (T.I.R.) pSS2_RBS = 250000 (T.I.R.) ( j( 0( @( )(( The2 native2 biosensor2 circuitA2 iOeO2 pNatSensA2 was2 constructed2 and2 its2 response2 curve2 characterizedO2 Wespite2the2interesting2and2useful2characteristics2of2this2 pNatSens2biosensorA2such2as2sensitivityA2negligible2leaky2 expression2and2relevant2operational2and2dynamic2rangeA2 the2 lack2 of2 any2 annotation2 of2 the2 intergenic2 promoter2 region2 prevents2 tailoring2 this2 biosensor2 circuit2 for2 custom2 applications2 in2 metabolic2 engineering2 strategiesO2 ThereforeA2 comparative2 sequence2 analysis2 was2 performed2 to2 identify2 engineering2 target2 such2 asA2 transcription2 factor2 binding2 sitesA2 transcription2 start2 sites2and2core2promoter2sequencesO Ligand2concentration2,mgFlw KluorescenceFoptical2density2,&w pSynSensPOX pSynSens)OX Hill2function
Transcript of Tailor-made transcriptional biosensors for optimizing ... · Tailor-made transcriptional biosensors...
Tailor-made transcriptional biosensors for optimizing microbial cell factories
BrechtGDeGPaepe1,GJoGMaertens1,GBartelGVanholme2GandGMarjanGDeGMey1
1CSBG-GCentreGforGSyntheticGBiologyG-GGhentGUniversityG-GBelgium2VIBG-GVlaamsGInstituutGvoorGBiotechnologieG-GDepartmentGofGPlantGSystemsGBiologyG-GBelgiumG
Centre forSynthetic Biology
pNatSens2&27haracterization2of2native2biosensor2circuit
pSynSens2&2Wecoupling2of2the2detector2and2effector2module
pSynSens)OX2&2àngineering2the2detector2module pSynSensPOX2&2àngineering2the2effector2module22
pNatSens
IntroductionMonitoring2the2intra&2and2extracellular2environment2and2adapting2metabolic2processes2accordingly2is2key2to2properly2address2the2enormous2complexity2of2today’s2metabolic2engineering2questionsO2To2
this2endA2scientists2are2reprogramming2nature’s2myriad2of2transcriptional2regulatory2systems2into2transcriptional2biosensors2which2are2able2to2detect2small2molecules2andA2in2responseA2express2output2
signalsO2In2this2mannerA2biosensors2propel2key2applicationsA2such2as2high&throughput2screeningA2adaptive2laboratory2evolution2and2dynamic2pathway2controlO2HenceA2transcriptional2biosensors2bear2
the2potential2to2augment2and2accelerate2current2metabolic2engineering2strategiesA2catalyzing2vital2advances2in2industrial2biotechnologyO2HoweverA2the2two2key2characteristics2of2these2natural2biosensor2
circuitsA2 the2response2curve2and2 ligand2specificityA2are2 typically2not2 in2 line2with2 the2requirements2 for2a2 real&life2biosensor2applicationO2 InsteadA2 they2are2evolutionary2optimized2for2specific2 in2vivo2
conditionsO27onsequentlyA2engineering2efforts2are2imperative2to2generate2tailor&made2biosensors2witch2custom2response2curves2and2ligand2specificitiesO2HenceA2in2this2contribution2to2address2these2
limitations2of2natural2biosensorsA2several2engineering2principles2were2established2to2create2biosensors2with2custom2response2curvesO2
The2authors2wish2to2thank2the2M8gentschap2Innoveren2q2OndernemenM2 in2Klanders2,8IOw2for2financial2
support2in2the2framework2of2the2PhOWO2grant2of2/recht2We2PaepeO2This2research2is2also2supported2by2the2
7ondex2project2,GO(BP)O)BNwO2
/recht2We2Paepe2&2/rechtOWePaepe4UGentObe
7entre2for2Synthetic2/iology2&2Ghent2University
7oupure2Links20GBA2U(((2Ghent2&2/elgium2
Wetector2module àffector2module
Transcription factor Fluorescent protein
Ligand2molecule
Kluorescent2proteinTranscription2factor Transcription2factor2binding2sites
Promoter R/S 7oding2sequence
pSynSens
8cknowledgments 7ontact
Wetector2module àffector2module
R/S2&2Promoter2&2Spacer2&2Terminator
?
Kitted2Hill2function2of2pNatSens
Response2curve2of2pNatSens
P(( j( 0( @( )((
Ligand2concentration2,mgFlw
Klu
ore
scenceFopti
cal2d
ensit
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&w
P(( j( 0( @( )((
Kitted2Hill2function2of2pSynSens
Response2curve2of2pSynSens
P(((
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j(((
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j(((
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B(((
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0(((
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5'UTR 5'UTR
With2 the2 different2 genetic2 parts2 identifiedA2 the2 path2 is2
paved2to2engineer2this2biosensor2circuit2by2altering2the2
expression2of2the2detector2and2effector2moduleA2vis&à&
vis2each2otherO2HoweverA2both2modules2are2entwined2in2
this2bidirectional2intergenic2architecture2and2can2not2be2
altered2 without2 affecting2 the2 opposite2 moduleO2
ThereforeA2the2two2modules2are2decoupled2by2inserting2
a2 terminator2 and2 spacer2 sequence2 together2 with2 a2
defined2 promoter2 and2 R/S2 sequence2 to2 constitutively2
express2 the2 transcription2 factorO2 This2 synthetic2
biosensor2 circuitA2 iOeO2 pSynSensA2 was2 constructed2
without2loss2of2functionality2in2comparison2to2pNatSensO
/y2varying2the2R/S2strength2of2the2detector2moduleA2a2set2of2custom2biosensors2was2created2with2
differing2 characteristics2 such2 asA2 increased2 operational2 and2 dynamic2 rangeA2 varying2 sensitivity2
and2cooperativityA2resulting2in2distinct2Hill2parametersO2NoteworthyA2when2targeting2the2detector2
moduleA2 specific2 R/S2 strengths2 resulted2 in2 increased2 output2 variability2 which2 is2 atributed2 to2
unbalanced2levels2of2transcription2factor2in2relation2to2the2regulatory2mechanismO2
8kin2to2the2detector2moduleA2varying2the2R/S2strength2of2the2effector2module2resulted2in2a2set2of2
custom2 biosensors2 with2 differing2 characteristicsO2 NoteworthyA2 when2 targeting2 the2 effector2
moduleA2 a2 wider2 range2 of2 maximum2 response2 levelsA2 with2 twofold2 higher2 maximum2 response2
levelsA2was2achieved2in2comparison2to2engineering2the2detector2moduleO
Ligand2concentration2,mgFlw
Klu
ore
scenceFopti
cal2d
ensit
y2,
&w
P(( j( 0( @( )(((
P(((
j(((
0(((
@(((
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)P(((
pSynSens FitpSS1_RBS = 10 (T.I.R.)pSS1_RBS = 100 (T.I.R.)pSS1_RBS = 500 (T.I.R.)pSS1_RBS = 1000 (T.I.R.)pSS1_RBS = 2500(T.I.R.)pSS1_RBS = 5000 (T.I.R.)pSS1_RBS = 20000 (T.I.R.)
pSynSenspSynSens
0
5000
10000
15000
20000
25000
0
20
40
60
80
100
0.0
0.5
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R/S2strength2,TOIOROw R/S2strength2,TOIORO2&2translation2initiation2ratew R/S2strength2,TOIOROw
pSynSens)(( G(( )((( PG(( G((( P((((
R/S2strength2,TOIOROw R/S2strength2,TOIORO2&2translation2initiation2ratew R/S2strength2,TOIOROw
25000
0
5000
10000
15000
20000
k2&
2Maxim
um
2resp
onse2level2,&
w
0
20
40
60
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100
pSynSens)(( G(( )((( PG(( G((( P(((()(((( )G((( PG(((
KM
2&2H
ill2consta
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n2&
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Klu
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ensit
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pSynSens FitpSS2_RBS = 10 (T.I.R.)pSS2_RBS = 100 (T.I.R.)pSS2_RBS = 500 (T.I.R.)pSS2_RBS = 1000 (T.I.R.)pSS2_RBS = 2500 (T.I.R.)pSS2_RBS = 5000 (T.I.R.)pSS2_RBS = 10000 (T.I.R.)pSS2_RBS = 15000 (T.I.R.)pSS2_RBS = 20000 (T.I.R.)pSS2_RBS = 250000 (T.I.R.)
( j( 0( @( )((
The2 native2 biosensor2 circuitA2 iOeO2 pNatSensA2 was2
constructed2 and2 its2 response2 curve2 characterizedO2
Wespite2the2interesting2and2useful2characteristics2of2this2
pNatSens2biosensorA2such2as2sensitivityA2negligible2leaky2
expression2and2relevant2operational2and2dynamic2rangeA2
the2 lack2 of2 any2 annotation2 of2 the2 intergenic2 promoter2
region2 prevents2 tailoring2 this2 biosensor2 circuit2 for2
custom2 applications2 in2 metabolic2 engineering2
strategiesO2 ThereforeA2 comparative2 sequence2 analysis2
was2 performed2 to2 identify2 engineering2 target2 such2 asA2
transcription2 factor2 binding2 sitesA2 transcription2 start2
sites2and2core2promoter2sequencesO Ligand2concentration2,mgFlw
Klu
ore
scenceFopti
cal2densit
y2,
&w
pSynSensPOXpSynSens)OX
Hill2function
