Zeng, An Ping

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Engineering Conferences InternationalECI Digital ArchivesMetabolic Engineering IX ProceedingsSummer 6-6-2012Structure-based Metabolic Engineering andSynthetic Biology for Efficient Strain DevelopmentAn Ping ZengTechnische Universitat Hamburg-HarburgFollow 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 CitationAn Ping Zeng, "Structure-based Metabolic Engineering and Synthetic Biology for Efficient Strain Development" in "MetabolicEngineering IX", E. Heinzle, Saarland Univ.; P. Soucaille, INSA; G. Whited, Danisco Eds, ECI Symposium Series, (2013).http://dc.engconfintl.org/metabolic_ix/13Structure-based metabolic engineering and synthetic biology for efficient    strain development An-Ping ZengInstitute of Bioprocess and Biosystems EngineeringHamburg University of TechnologyOur research areas: 1.Design of biocatalysts2.Systems Biology / Modeling 3.Integrated bioprocesses / BiorefineryPresent praxis of metabolic engineering and its limitation in strain developmentSubstrateSin M1 PinIdentify rate- Manipulate genes EnzymesFeedbackinhibitionProductlimiting stepor pathwayEvaluate strain(expression & deletion) Not efficient for protein-level limitationOften Yes/No changes/lack of dynamic controlMostly trial and errorDesign and control of bioreactions at atomic level and from first principle4Make use of the potentials in structural biology and computingWorld’s most brilliant X-ray and light sources for structural study at DESY/HamburgBeyond the static structure 5Petra III European free-electron laser facilitiesalso „see“ dynamics at fs (10-15s)!  Parts                                         (Proteins, domains etc.)Structural metabolic engineering and Structural modeling       and designDesigned cellModules (Designed  protein/complex )Functional assemblies (Synthetic pathways)Device for reprogramming         networksynthetic biologyNovel protein as biocatalyst or drugs Example: Strain development for amino acid productionCorynebacterium glutamicum Lysine: > 1.5 Mio. t/yE. coliProblem: Allosteric feedback inhibitionExample: Aspartokinase (lysC) catalyse the first and limiting   step of synthesis of aspartate  family amino acidsGlucose8 LysineAllosteric inhibition   A general problem limiting product yield and titer in many industrial bioprocesses!How to combat allosteric inhibition?LysineTraditional view of allostery: Binding of an effector and conformational change of the enzymeSolution:Mutation of the enzyme to avoid effector binding !Random mutation over 50 years identified 6 (reported) targets for lysC Random Mutation: e.g. UV-Light, X-ray Selection:  Analoge Chemicals, AuxotrophicTime-consuming and many other disadvantages!PatentingConventional approach identified mainly mutations on the protein surface xxxInhibitorxxOur new approach:To identify and modify the signal transduction pathway xxxSubstrateKey: To find the residue-residue interaction networkof conformational changeZ. Chen et al. Appl. Environ. Microbiol. 2011; Z. Chen et al. J. Biotechnol 2011Targeted molecular dynamic simulationConformational change of lysC upon binding of lysine 50100150200250300350 0.5Mapping the patterns of atomic motionto dynamic residue interaction networkMD withouteffector  50 100 150 200 250 300 350 40050100150200250300350400-0.500.5 50 100 150 200 250 300 350 400400-0.50Correlated coefficient matrixMD witheffectorCo-evolutionary analysis is also  very useful to study allostery 44789131174Correlated mutation matrixMultiple sequences alignment 4 47 89 131 174 216 256 297 337 385 4262162562973373854260123Conservation of residues Correlated mutations-Structurally or functionally relevant-Adopted to import new function or regulationS315H320V347E346S345T344R416N414F405M318Evolutionary and MD analyses revealed signaling network of AK3 at atomic levelTargetsAll the previous 6 mutations from the last decades also found!M251T253R305I392V339S338 I337T355T352F329L325F324G323V349M417N426C428S423S424 C378Z. Chen et al. J. Biotechnol. 2011 30 mutations predicted and experimentally confirmed with reduced feedback inhibition0.811.2Relative activityQ308AwildtypeM251PT253RR305AR305DE. coli AK3 mutants00.20.40.60 4 8 12 16 20Relative activityLysine concentration(mM)S315AH320AF329RI337PS338LV339AE346RR416AC428RWildtype1.2 Wild-typePredictions also applied to aspartokinase from C. glutamicum1.4 Mutant N372A00.20.40.60.810 5 10 15Inhibitor concentration (mM)+Lysine+Threonine+Lysine+Threonine (1:1)00.20.40.60.811.20 5 10 15Inhibitor concentration (mM)+Lysine+Threonine+Lysine+Threonine(1:1)Relative activityWildtype vs. point mutation of lysC for lysine production in C. glutamicum80100120140OD66030405060Lysine(g/L)No negative effect  on 02040600 5 10 15 20 25 30OD660Time (h)wildtypeLC298010200 5 10 15 20 25 30Lysine(g/L)Time (h)wildtypeLC298growth!From „zero to hero“ with a single mutation and in a minimal medium6080100120LysineCDWConcentration (g/L)19020400 10 20 30 40 50 60Fermentation time (h)Concentration (g/L)Systematic rational design of pathwaysEnhance precursor availabilityG35T36K37NADPIncrease NADPH supplyOn the way beyond 150 g/L by design in short time!Chen et al. J. Biotechnol. 2011; AEM. 2011; Geng et al. AMB, 2012,  Zeng et al. Patent applicationsR441E443E417T365E240Y238R281L286Reengineering of threonine deaminase for theronine production in E.coli21L. Chen et al. Appl Microb Biotechnol 2012 ACT ACTThreonine(B)Redesign of homoserine dehydrogenaseNatively inhibited by threonineDesign based on predicted allosteric signal transduction R441E443E417T365E240Y238R281L286between catalytic domain and regulatory domainSubstrate(Glucose)MCell growth/ByproductsSynthetic protein for dynamic and self-regulation of biosynthesis Needed only in the cell growth phase of a fermentationhom ThrProduct(lysine)Intracellularproduct as signalArtificial allosteric controlPoster 4 . Chen et al.Modular design of artificial enzymeSensor domainControl domain Redesign of signal molecule binding sites in the sensor domain Identify the (allosteric) signaling pathway Protein-protein docking to predict domain-domain interaction mode Design of specific linker to provide potential domain interface Combination of rational design with directed evolutionInterfaceNew concept for protein design: From structure via dynamic modules to functionEquilibrium DynamicsNon-equilibrium DynamicsConformation Energy TimeEnergyC. Ma. PLoS ONE 6(10): e26453. 2011C.Ma. PLos One 7(2):e31529, 2012C. Ma et al. Submitted. 2012 Distribution DissipationMD simulationPoster 191 Ma and Zeng. New Concept and Algorithms to Reveal Protein Dynamics and Intramolecular Interaction Network and Their Application for Protein Design Energy dissipation of key residues in AK326From structural to dynamic modules revealed by the new method27Synthetic AK3 for response todifferent effectorsDesign of non-native lysine responsive HSD• The integration of MD and evolutionary analysis is a powerful and predictive appraoch for the re-design of allosteric regulation of enzymes of amino acid systhesis.– For three different aspartokinases from E.coli and C. glutamicum30 efficient mutation points have been successfully identified and experimenally verified.Summary• Systematic structure-based design led to highly productive strain of L-lysine in a very short time.   • Synthetic protein designed for dynamic and self-regulation of biosynthesis – New concept and algorithms for protein dynamics developed• Structure-based design provides new possibilities for a rational development of biocatalysts.     30S B CSPSubstrateProduct4. Transport process3. Signaling process EffectorPerspective: Structural ME and SynBio  SScaffold proteinP2. Enzymes        & pathway e.g. allosteric regulation Regulator1. Genetic circuitsDNARNA(e.g. riboswitch)Chen et al. (2010) TIB, 28, 534ThAcknowledgementLin ChenZhen ChenDonita FrankFeng GengChengwei MaDr. Sugima RappertFunding by EU, BMBF, DFG, Hamburg excellence cluster SynBio

Structure-based Metabolic Engineering and Synthetic Biology for Efficient Strain Development

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Structure-based Metabolic Engineering and Synthetic Biology for Efficient Strain Development

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