iad 824: An Improved Genome- scale Metabolic Network Model for Scheffersomyces S,pi,s

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1 1 iad 824: An Improved Genome- scale Metabolic Network Model for Scheffersomyces S,pi,s Andrew Damiani 1, Q. Peter He 2, Thomas Jeffries 3, Jin Wang 1 1. Department of Chemical Engineering, Auburn University, Auburn, AL 2. Department of Chemical Engineering, Tuskegee University, Tuskegee, AL 3. Department of Bacteriology, University of Wisconsin- Madison, Madison, WI Auburn Chemical Engineering

2 Overview Introduc+on MoXvaXon System idenxficaxon framework (SID) Previous work on genome- scale models Modified Model Background Example SID Valida+on Experiments QualitaXve and QuanXtaXve SID experiments Xylitol producxon: cofactor balance Conclusion 2

3 Why model metabolic networks? 3

4 The associaxon of metabolism with genome Lewis, Nagarajan and Palsson, Nature Reviews Microbiology, 10, 291,

5 ReconstrucXon of Genome- Scale Models Osterlund, Nookaew and Nielsen, Biotechnology Advances, 30, 979,

Gaps to fill Difficult to validate model and assess model accuracy due to the scale and complexity Difficult to extract mechanisxc insight embedded in the metabolic

6 Gaps to fill Difficult to validate model and assess model accuracy due to the scale and complexity Difficult to extract mechanisxc insight embedded in the metabolic network from numerical simulaxon results In other words, how to obtain comprehensive, qualitaxve understandings from the oeen complicated quanxtaxve computaxonal results? 6

7 What is system idenxficaxon based framework? 7

8 Systems idenxficaxon based framework Design Experiments Black- Box Genome- scale Model in silico Results System ID to Extract Biological InformaXon Knowledge *Liang, Damiani, He & Wang, ACS Sustainable chemistry & Engineering, 2, 38,

9 Example: Systems idenxficaxon based framework Relative Ethanol Loading Ethanol 10 5 Relative Loading rx1 rx2 rx3 rx4 rx5 2 rx6 rx7 rx8 4 rx9 rx10 rx Oxygen uptake Reactions Xylose uptake rx1 rx2 rx3 rx4 0 2rx5 rx6 rx7 rx8 rx9 rx10 rx11 Oxygen uptake Reactions Xylose uptake 9

naxve capacity for xylose fermentaxon Role of redox balance in xylose

10 Model system: Scheffersomyces sxpixs Scheffersomyces s,pi,s is very important for biofuels producxon from lignocellulosic biomass Highest naxve capacity for xylose fermentaxon Role of redox balance in xylose metabolism of S. s,pi,s SensiXvity to oxygen uptake driven by redox balance 10

11 Metabolic network models for S. s,pi,s Two genome- scale models have been published on S. s,p,ts iss884: Caspeta et al. BMC System Biology, 2012, 6:24 ibb814: Balagurunathan et al. Microbial Cell Factories, 2012, 11:27 Specifica+on iss884 ibb814 Approach Semi- AutomaXc Manual Reac+ons Cytosol Mitochondria Peroxisome 60 N/A Transport Metabolites Genes

12 Recap and limitaxons of metabolic network models Purpose iss884 ibb814 QualitaXve Experiments QuanXtaXve Experiments Sys. ID Experiments Metabolic Flow Xylitol ProducXon 12

13 Design of in silico experiments Basic idea: examine how the models respond to increased oxygen uptake rate (OUR) in oxygen limited condixon. The correct answer is known: increase fluxes through TCA cycle, Electron Transport Chain, and as well as increased fluxed through Pentose Phosphate Pathway and Glycolysis. If the system ID results match the correct answer, then the model is accurate, at least in this aspect. 13

14 In silico experimental setup Xylose pick up rate: upper limit at 5 mmol/gdw*hr; Two oxygen pick up rate ranges are examined; Oxygen- limited : mmol/gdw*hr Ammonium, sulphate, and phosphate: unconstrained Color code (Traffic light) Red decrease Green increase Gray neutral 14

15 System ID Results Oxygen limited iss884 ibb814 15

16 UXlizaXon of System idenxficaxon framework Aerobic condixons: OxidaXve branch of Pentose Phosphate Pathway was not ac+ve 16

17 Major modificaxons 17

18 Modified Model Name: iad824, 1384 reacxons Based on ibb814 Our System idenxficaxon method was used to pinpoint where changes needed to be made Remove: 6 reacxons Add: 12 reacxons Change: 15 reacxons 18

19 System ID Results Oxygen limited iad824 ibb815 19

20 QuanXtaXve validaxon* Xylose Glucose micro- aerobic Xylose Glucose aerobic *Data from Li, DissertaXon, The University of Toronto,

21 QualitaXve validaxon- 1* *Experimental results from Jin, Cruz & Jeffries, Appl Microbiol Biot,

22 QualitaXve validaxon- 2* Run in silico simulaxons compare: Original vs. DeleXon Complete inhibixon, ParXal inhibixon,negligible,increase Complex I III AOX IV Effect on Growth Glucose Xylose Exp iad824 iss884 ibb814 Exp iad824 iss884 ibb814 NA IV and AOX I + del AOX I + del IV *Experimental results from Balagurunathan et al., Microb Cell Fact,

Xylitol producxon through cofactor manipulaxon Case # Ethanol Yield Experiment Ethanol Yield Predic+on Xylitol Experimental Xylitol Predic+on Ra+o of XR NADPH/NADH OUR Case 1 0.3277 0.3301 0.142 0.

23 Xylitol producxon through cofactor manipulaxon Case # Ethanol Yield Experiment Ethanol Yield Predic+on Xylitol Experimental Xylitol Predic+on Ra+o of XR NADPH/NADH OUR Case / Case / Case / Case / *Experimental results from Farias et al., Applied Biochemistry and Biotechnology,

24 Conclusion System IdenXficaXon based Framework is a powerful method to uncover biological essenxal informaxon iad824 is a superior model to the published models 24

25 Acknowledgement Financial support Sun Grant NSF , ; NSF - IGERT 25

26 QuesXons? 26

NIH Public Access Author Manuscript Nat Biotechnol. Author manuscript; available in PMC 2011 June 6.

NIH Public Access Author Manuscript Published in final edited form as: Nat Biotechnol. 2010 March ; 28(3): 245 248. doi:10.1038/nbt.1614. What is flux balance analysis? Jeffrey D. Orth, University of California