Eliasson Lantz, Anna

Nandy, Subir Kumar

Rattleff, Stig

Thykær, Jette

To improve the expression and secretion of heterologous proteins in the filamentous grampositive bacteria, Streptomyces lividans, bioprocessing strategies were developed. A mathematical model was constructed to study the effects of the process kinetic parameters on the production of the model-protein RFP. The model describes the dynamics of glucose consumption and formation of biomass in addition to RFP production. The model was also extended to describe a two substrate condition

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Downloaded from orbit.dtu.dk on: Dec 18, 2017Effect of kinetic parameters on heterologous protein production: a sysbio approachNandy, Subir Kumar; Rattleff, Stig; Thykær, Jette; Eliasson Lantz, AnnaPublication date:2011Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Nandy, S. K., Rattleff, S., Thykær, J., & Eliasson Lantz, A. (2011). Effect of kinetic parameters on heterologousprotein production: a sysbio approach. Poster session presented at 6th Danish Conference on Biotechnologyand Molecular Biology, Vejle, Denmark.Effect of kinetic parameters on heterologous protein production: a sysbio approachSubir Kumar Nandy1, Stig Rattleff1, Jette Thykær, Anna Eliasson Lantz*Summary: To improve the expression and secretion of heterologous proteins in the filamentous gram-positive bacteria, Streptomyces lividans, bioprocessing strategies were developed. A mathematicalmodel was constructed to study the effects of the process kinetic parameters on the production of themodel-protein RFP. The model describes the dynamics of glucose consumption and formation ofbiomass in addition to RFP production. The model was also extended to describe a two substrate1 Both the author have contributed equally to this work. * Corresponding Author: ael@bio.dtu.dkCenter for Microbial BiotechnologyDepartment of Systems BiologyResultsFigure 1: The single substrate kinetic model afteroptimization. Kinetic parameters are shown below.Lines are predictions and dots experimental data.µm: 0.2, (h-1), Km: 1.35 (g/L) , YSX: 0.37. The model isvalidated with different initial glucose concentrationsexample, 20, 30 and 40 g/L.Background Due to it´s ability to secrete proteins and low proteasecontent, Streptomyces have been considered as analternative host organism for producing recombinantproteins. In addition to genetic strategies, bioprocessingstrategies are also important for improved production ofprotein secretion from Streptomyces. In this work, a mathematical kinetic model of thesystem for RFP production was developed. Laboratorydata from batch fermentations involving growth andprotein production by S. lividans on a single substrate,glucose was used to estimate parameters in the model.The model was then also extended to describe growthand production on double substrates, glucose andglutamate.ObjectiveTo develop a kinetic model for the prediction of proteinproduction from S. lividans.condition.Schematic representation of processProtease assay: A2382 Sigma kit is used for this assay,Fluorescense assay: 530/25 excitation and 600/25emission, measured from biotek-plate reader.Spore stock1X108 spore cells / lit of total volume2YT medium30°C / 150 rpmCentrifuge10,000 rpm / 10 minDiscardsupernatantReconstitute withDH2O, sterilizedInnoculationANALYSIS    FOR    RFP    PRODUCTION  PROCESSODDCWTimeDCW % CO2∞∞HPLCPROTEASE ASSAYRFP ASSAYX  30 10 20 30 40 50 600510Glc, S (g/L)hrs0 10 20 30 40 50 600246Biomass, X (g/L)hrs0 10 20 30 40 50 6000.020.040.060.08Protease, Pr (g/L)hrs0 10 20 30 40 50 6000.050.1RFP (U/L)hrsGlc, S, (g/L)X, (g/L)Pr, (g/L)RFP, (U/L)0 10 20 30 40 50 600510Glc, S1 (g/L)hrs0 10 20 30 40 50 6002.55GTM, S2 (g/L)hrs24Biomass, X (g/L)Glc, S1, (g/L)X, (g/L)Glu, S2, (g/L)Figure 2: The double substrate kinetic model as avisualization. Kinetic parameters are: µm1: 0.2, (h-1), Km:1.35 (g/L) , YSX: 0.37, µm2: 0.1, (h-1), Km1: 1. 5 (g/L) , Km2:1.5. The plan for near future is to validate this modelwith different initial substrate concentrations forexample, Glc/Glu 20/10, 30/15 and 40/20 g/L.The Model Growth Rate…………. (1)µ: specific growth rate, (h-1),S: Substrate concentration, (g/L),µm: max. specific growth rate, (h-1),Km: Monod constant (g/L) . Temperature Effect…………… (2)Kd: Specific death rate, (h-1) Nutrient consumption and Product formationSubstrate = Substrate - Growth - Product - Maint.accumulation Feed Formation Reqd.- m*X…………. (3)YSX: Biomass yield on glucose Product FormationProduct accumulation = Formation – Destruction…… (4)....................................…. (5)…. (6)…. (7)Where,rP: Specific production rate of protease,rG: Specific production rate of GFP,KdG: GFP degradation rate by protease,rP=(αP*µ + βP)*(KSP/(KSP + S)) and rG=(αG*µ + βG)Materials and MethodsS. lividans TK24 was used throughout the study.The medium used in this study is (/L): NaH2PO4.H2O (1.38g),NH4Cl (2.675g), KCl (0.7455 g), Na2SO4 (0.284 g), Citric acid(0.42 g), MgCl2.6H2O (0.254 g), CaCl2.2H2O (0.184 g), Tracemetal solution (5ml), Antifoam (sigma 204, 0.2 ml), Vitamin(1ml), Glucose.H2O (10/20/30/40 g).ConclusionsComparison with experimental data demonstrated thatthe model is able to accurately predict the proteinproduction process kinetics. The model will be furtherapplied to simulate different process conditions in orderto gain a better understanding of the influence ofdifferent parameters on protein production.0 10 20 30 40 50 600Biomass, X (g/L)hrs0 10 20 30 40 50 600.010.0110.012Protease, Pr (g/L)hrs0 10 20 30 40 50 6000.20.4GFP/RFP (U/L)hrsX, (g/L)Pr, (g/L)RFP, (U/L)YSXYSX YSPCenter for Microbial BiotechnologyDepartment of Systems Biology

Effect of kinetic parameters on heterologous protein production: a sysbio approach

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Effect of kinetic parameters on heterologous protein production: a sysbio approach

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