Alper, Hal

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Engineering Conferences InternationalECI Digital ArchivesMetabolic Engineering IX ProceedingsSummer 6-6-2012Synthetic Control of Transcription: From HybridPromoters to Promoter Engineering to SyntheticOperon DesignHal AlperThe University of Texas at AustinFollow this and additional works at: http://dc.engconfintl.org/metabolic_ixPart of the Biomedical Engineering and Bioengineering CommonsThis Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion inMetabolic Engineering IX by an authorized administrator of ECI Digital Archives. For more information, please contact franco@bepress.com.Recommended CitationHal Alper, "Synthetic Control of Transcription: From Hybrid Promoters to Promoter Engineering to Synthetic Operon Design" in"Metabolic Engineering IX", E. Heinzle, Saarland Univ.; P. Soucaille, INSA; G. Whited, Danisco Eds, ECI Symposium Series, (2013).http://dc.engconfintl.org/metabolic_ix/15Synthetic control of transcription:From hybrid promoters to promoter engineering to synthetic operon designSeptember 7, 2005Hal AlperDepartment of Chemical EngineeringThe University of Texas at Austinhalper@che.utexas.eduhttp://www.che.utexas.edu/alper_group/1Metabolic Engineering has opened up possibilities…• Metabolic engineering advances have expanded the breath of chemicals produced by cellsLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper– Pharmaceuticals and Nutraceuticals– Fuels– Commodity/Specialty Chemicals– Polymers and Precursors2Curran & Alper, Metabolic Engineering, 2012…but, requires synthetic control elements...Pathway Control Circuit DesignABC DLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 3Heterologous Expression Probing via Graded Expressionpromoterpromoterpromoterpromoter HATHAT*ABCD…with a range of expression capacities.Native range of mRNA levelspromoterpromoterRequire a promoter series(s) that span above the highest native expression down to essentially zero expression:Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 4Holstege, et al. (1998), Cell 95:717-728.promoterpromoterAffords both amplification and knockdowns.Synthetic control of transcription occurs at many levelsSyntheticTranscriptional Amplifiers5’UTR and MCSsGenetic ContextElements that Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 5Mutant PromotersSelection Markersand VectorsInfluence Expression Terminators and 3’UTRDesigning novel genetic control elements• Promoter Engineering is an effective method for generating a collection of genetic control elementsPrior Promoter engineering exampleLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 6Alper et al. PNAS, 2005.GFP+Promoter PromoterLibraryRange of promoter strength to control gene expressionDevelopment of diversified promotersyECitrineTCYC1Mutant PromoterLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 7Nevoigt et al. AEM, 2006.Development of novel low-strength promoters2.533.54Fluorescence Relative to TFC1yECitrineTCYC1Mutant PromoterLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 800.511.52Fluorescence Relative to TFC1PromoterAn expanding range of yeast promotersNative range of mRNA levelsLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 9Holstege, et al. (1998), Cell 95:717-728.Mutant Promoter Libraries• Promoter engineering via random mutagenesis is effective at creating diversity• Bias in promoter libraries toward lower expression capacityLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper• Expression range nearly matches “native expression levels”—need more sequence diversity• Require novel tools for increasing strength of promoters10Synthetic control of transcription occurs at many levelsSyntheticTranscriptional Amplifiers5’UTR and MCSsGenetic ContextElements that Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 11Mutant PromotersSelection Markersand VectorsInfluence Expression Terminators and 3’UTRPromoter Engineering in Yarrowia lipolyticaYarrowia lipolytica is a fully sequenced, oleaginous yeast• Naturally accumulates fatty acids (including linoleic acid) on food source with high C:N ratio Issues with immature genetic tools and expression levelsAdvantages ChallengesLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 12• Semi-developed genetic tools• Thrives on non-conventional carbon sources Low plasmid copy number Lack of genetic tools “Strong” promoters do not readily existY. lipolytica Promoter Engineering via tandem UAS sequencesPrior research suggested linking upstream activating sequences (UAS) to a minimal promoter can create function.  Analyzed 1 to 4 UAS site.Madzak et al., Microbiology, 1999UAS1b UAS1b UAS1b UAS1b pleumLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 13UAS1b pleum GFP… UAS1b1 nWe sought to evaluate potential of modulating promoter activity by tandem copies of UAS1b sites (n = 1 to 32)Creation of a strong promoter set in Y. lipolytica200250300350400450500Mean FluorescenceLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 14050100150Cen1TEF(UAS1b)1(UAS1b)2(UAS1b)3(UAS1b)4(UAS1b)5(UAS1b)6(UAS1b)7(UAS1b)8(UAS1b)9(UAS1b)10(UAS1b)11(UAS1b)12(UAS1b)13(UAS1b)14(UAS1b)15(UAS1b)16(UAS1b)17(UAS1b)18(UAS1b)19(UAS1b)20(UAS1b)21(UAS1b)22(UAS1b)23(UAS1b)24(UAS1b)25(UAS1b)26Promoter ConstructMean FluorescenceBlazeck et al., AEM, 77(22), 2011Extension of hybrid promoter strategypleum GFPUAS1b16UAS16 Enabled TEF Promoters Tuning450500Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 15TEF promoter truncation series050100150200250300350400LEUM TEFS1 TEFS2 TEFS3 TEF TEFL1 TEFL2 TEFL3 TEFL4Basal PromoterMean Fluorescence Basal PromoterUAS16 BasalPromoterIdentification and use of a TEF UASLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 16Blazeck et al., Submitted, AMB, 2012Expanding the hybrid promoter approach to S. cerevisiae150002000025000FluorescenceCit UASClb UASCYCTEFXUAS Elements Core PromotersLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 170500010000FluorescencePromoterTef UASGu1 UASGPDGALBlazeck et al., Accepted/In Press, Biotech Bioeng, 2012Creating the strongest constitutive promoter150002000025000FluorescenceHybrid promoter approach created a promoter 60% higher on fluorescence and 2.5 fold higher in mRNA Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 180500010000p416MCS Gpd GpdClbCittefsc3FluorescencePromoterthan the strongest constitutive promoter by mixing disparate UAS elementsBlazeck et al., Accepted/In Press, Biotech Bioeng, 2012Creating a range of inducible hybrid promotersGalactose –Inducible Hybrid PromotersGal4pBS1-PLEUMGal4pBS1 PLEUM++Gal4p Binding Sites PLEUM CoreCGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGGal4pBS1 Gal4pBS3 Gal4pBS4Gal4pBS2Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 19PLEUMPLEUMPLEUMPLEUMGal4pBS2-PLEUMGal4pBS3-PLEUMGal4pBS4-PLEUMGal4pBS12-PLEUMPLEUM Gal4pBS13-PLEUMGal4pBS24-PLEUMGal4pBS34-PLEUMGal4pBS134-PLEUM+++++++Gal4pBS2PLEUMPLEUMPLEUMGal4pBS1Gal4pBS3Gal4pBS4Gal4pBS2Gal4pBS1 Gal4pBS3Gal4pBS2 Gal4pBS4Gal4pBS3 Gal4pBS4Gal4pBS3 Gal4pBS4Gal4pBS1Creating a range of inducible hybrid promoters30000400005000060000Mean Fluorescence (RFU)GalactoseGlucoseLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 2001000020000Mean Fluorescence (RFU)PromoterBlazeck et al., Accepted/In Press, Biotech Bioeng, 2012Hybrid Promoter Engineering ExamplesLargest library of promoters in Y. lipolytica Strongest constitutive promoter in S. cerevisiaeLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 21Largest range of synthetic inducible promoters in S. cerevisiaeBlazeck et al., AEM, 77(22), 2011 & Blazeck et al., Accepted/In Press, Biotech Bioeng, 2012Synthetic, hybrid promoters• UAS elements serve as synthetic transcriptional amplifiers• UAS elements and core promoters can serve as modular synthetic componentsLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper• Hybrid promoter engineering can amplify expression and create highly-functional libraries22Synthetic control of transcription occurs at many levelsSyntheticTranscriptional Amplifiers5’UTR and MCSsGenetic ContextElements that Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 23Mutant PromotersSelection Markersand VectorsInfluence Expression Terminators and 3’UTRGOIMulticloning sites influence 5’UTR sequencesLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 24Promoter RS1   RS2  RS3    RS4     RS5       RS6              Crook et al., Nucleic Acids Research, 39(14), e92, 2011Model System• Yeast model system (S. cerevisiae)• pBluescript SK II Multicloning Site• CYC1, TEF, or GPD promotersLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper• yECitrine– Codon-optimized25>80%3-foldRestriction site affects protein expression Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 26absolutevariationrelativevariationEffect is most pronounced with short, codon-optimized genesNormalized Reporter ExpressionLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 27Normalized Reporter ExpressionModeling and re-designing MCSs• Goal: Use model to re-design better MCSs not susceptible to position effect by mitigating 5’UTR secondary structureLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 28NNNNNConceptual model of inhibition• Region(s) of secondary structure in 5’UTR can impede ribosome progressionLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 29Modeling and re-designing MCSs:GPD/TDH3-based PromoterLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 30Crook et al., Nucleic Acids Research, 39(14), e92, 20115’UTR Influence• Choice of site in MCS can influence 5’UTR regions via secondary structure• Most pronounced by short, codon-optimized genes (i.e. when transcription Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alperand translation rates are not limiting)• Can use model-based approach to improve MCS design (esp. with GPD)• Genetic context is important for characterizing parts31Synthetic control of transcription occurs at many levelsSyntheticTranscriptional Amplifiers5’UTR and MCSsGenetic ContextElements that Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 32Mutant PromotersSelection Markersand VectorsInfluence Expression Terminators and 3’UTRImportance of genetic contextLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 33Importance of genetic contextLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 34Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 35Genetic Context• Results highlight importance of genetic context of synthetic parts– Plasmids vs. genomic integration site• Highlight need for insulating elementsLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 36Synthetic control of transcription occurs at many levelsSyntheticTranscriptional Amplifiers5’UTR and MCSsGenetic ContextElements that Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 37Mutant PromotersSelection Markersand VectorsInfluence Expression Terminators and 3’UTRTerminator choice can influence performanceTest impact of changing terminator regionLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 38>5 fold difference in expression output by changing terminatorSynthetic control of transcription occurs at many levelsSyntheticTranscriptional Amplifiers5’UTR and MCSsGenetic ContextElements that Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 39Mutant PromotersSelection Markersand VectorsInfluence Expression Terminators and 3’UTRFinal Thoughts• Synthetic control elements are a critical tool for implementing M.E. strategy• “Promoters” cannot be thought of as singular, isolated elements• Currently require slightly more range, but Laboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alpersignificantly more sequence diversity• Starting to understanding fundamental design principles for this critical components• These elements are greatly enhancing our capacity to metabolically engineer pathways40AcknowledgementsStudentsGraduateJohn BlazeckJoseph ChengNathan CrookKate CurranAmanda LanzaJohn LeavittSun-mi LeeLeqian LiuUndergraduateVaibhav AgarwalaHeming BaiAlex BaileyAustin ComerTim DyessRishi GargRachelle GerstnerAkash GuptaLaboratory for Cellularand Metabolic EngineeringJune 6, 2012Slide #     Hal Alper 41Heidi ReddenEric YoungDaniel HuangTaylor JellisonAshty KarimDo Soon KimPeter OtoupalAshley PoucherAnnie PanHeidi ReddenBen ReedLindsey ReyAndrea Zuzack

Synthetic Control of Transcription: From Hybrid Promoters to Promoter Engineering to Synthetic Operon Design

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Synthetic Control of Transcription: From Hybrid Promoters to Promoter Engineering to Synthetic Operon Design

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